Thin porous layers for thin-layer chromatography

ABSTRACT

Novel separation materials for thin layer chromatography are disclosed; these consist of a support and a separation medium layer and are obtainable by the following process steps: 
     a) cleaning of the support; 
     b) application of a liquid film comprising a polysilicic ester to a support; 
     c) introduction of the support with the liquid film into an atmosphere which effects hydrolysis and further polymerization of the polysilicic ester; 
     d) hydrolysis and further polymerization of the polysilicic ester at constant temperature; 
     e) washing of the silica layer; 
     f) drying of the silica layer. 
     The silica gel surface of the separation material of the invention can be modified by means of separation effectors.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/EP99/00284 filed Jan. 19, 1999.

The invention relates to separation materials for planar chromatographywhich have porous separation medium layers of low thickness and aresuitable for the separation of substances on the nL scale.

Increasing demands made of the performance of analytical methods, inparticular in respect of speed and detection sensitivity, mean that inthe case of thin layer chromatography (planar chromatography) there is aneed for separation medium layers which are significantly thinner thanthose obtainable hitherto, i.e. thinner than 50 μm.

Cremer and Nau (1968) describe experiments (Natur-wissenschaften 55,page 651) in which chromatographic separations were carried out onultrathin layers. The layers used consisted of indium oxide, bismuthoxide or tin oxide and had a thickness of 1 μm or less. These layerswere vapour-deposited. The substances were able to be separated on modelsystems, but the method has not found further use. For thin layerchromatography, the practice has thus hitherto been to apply particulateseparation media, which can have spherical or irregular shapes, to asupport. In this procedure, it is possible to add auxiliaries such asbinders, fluorescent indicators, reflection reinforcers or the like tothe layer. Such additives are well known to those skilled in the art. Inthis method, it does not appear to be possible to reduce the thicknessof the separation medium layer to less than 50 μm.

It has been found that significantly thinner separation medium layers,for example layers having a thickness of about 10 μm, can be producedusing a completely different principle which has hitherto not beencustomary for the production of separation medium layers of silica gelfor thin layer chromatography.

These processes and the separation materials for thin layerchromatography produced by means of these processes are provided by thepresent invention.

DE 19 08 695 discloses methods of applying thin coherent films oftitanium dioxide and/or zirconium dioxide having a thickness of lessthan 1 μm to plastic films. This is said to prevent impurities from theplastic getting into the separation medium layer. However, these layersof titanium dioxide and/or zirconium dioxide have no chromatographicactivity.

The invention provides separation materials for thin layerchromatography consisting of a support and a separation medium layerobtainable by the following process steps:

a) cleaning of the support;

b) application of a liquid film comprising a polysilicic ester to asupport;

c) introduction of the support with the liquid film into an atmospherewhich effects hydrolysis and further polymerization of the polysilicicester;

d) hydrolysis and further polymerization of the polysilicic ester atconstant temperature;

e) washing of the silica layer;

f) drying of the silica layer.

The invention further provides separation materials having the featuresmentioned, whose silica gel surface is modified by means of separationeffectors.

The invention also provides for the use of separation materials havingthe features mentioned for separation of at least two substances by thinlayer chromatography.

Finally, the invention provides a process for producing separationmaterials, which comprises the following steps:

a) cleaning of the support;

b) application of a liquid film comprising a polysilicic ester to asupport;

c) introduction of the support with the liquid film into an atmospherewhich effects hydrolysis and further polymerization of the polysilicicester;

d) hydrolysis and further polymerization of the polysilicic ester atconstant temperature;

e) washing of the silica layer;

f) drying of the silica layer;

where, in preferred embodiments, the process step b) is carried out byspraying the solution onto the support or the liquid film is applied byfirstly applying the liquid at one point and subsequently rotating thesupport around the point of application, with the axis of rotationperpendicular to the plane of the support, or by distributing the liquidfilm using a roller.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 schematically shows the separation of a dye mixture.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 schematically shows, in enlarged form, the separation of a dyemixture. The separated dyes A, B and C are shown on four tracks on theseparation material (1); (2) marks the position of the eluant front. Theoriginal size of the separation material is about 14×56 mm; furtherexperimental details are given in the description (Use Example A).

For the purposes of the present invention, the term support refersessentially to the supports customary in thin layer chromatography, forexample plastic films or metal foils and in particular glass plates.However, owing to the miniaturization of the separation materials of theinvention, it is in principle also possible to use other supports whichhave hitherto not been used in customary thin layer materials, forexample for cost reasons; conceivable supports are silicon wafers orfused quartz.

The chromatographic separation takes place in the separation mediumlayer. In the prior art, the separation medium layer in separationmaterials for thin layer chromatography comprises, for example,particulate silica gel which may be modified by means of separationeffectors and possibly further additives such as binders, fluorescentdyes or reflection reinforcers. The separation medium layers of theinvention comprise silica gel which is produced in situ from polysilicicesters by hydrolysis and polymerization. The separation layers of theinvention may further comprise customary additives; for example they canbe modified by means of separation effectors or, for example, afluorescence indicator can be incorporated into the separation mediumlayer.

The alcohol component of the polysilicic esters used according to theinvention is derived from C₁-C₅-alcohols, in particular methanol,ethanol, n-propanol or i-propanol. The mean molecular weight of thepolysilicic esters used according to the invention is in the range from500 to 2000, preferably from 800 to 1500. Such polysilicic esters arecommercially available. The polysilicic esters used according to theinvention can be used as pure substances or as mixtures having variousmolecular weights and/or different alcohol components. It is alsopossible to dilute the polysilicic esters used according to theinvention with suitable solvents; solvents which have been found to beparticular useful are C₁-C₅-alcohols, for example methanol, ethanol,n-propanol or i-propanol. The solvent is preferably chosen so that thesolvent and the alcohol component of the polysilicic ester are the same.The polysilicic ester content of the diluted solution should be at least30% by weight.

The cleaning of the support is an important first process step. Here,the use of sodium hydroxide solution and detergents (e.g. Extran®) hasbeen found to be useful; before the next step, the support is rinsedthoroughly with water.

The polysilicic ester subsequently has to be applied as a uniform liquidfilm to the support. Two methods have been found to be particularlyuseful for this purpose:

a) A sufficient liquid volume comprising the polysilicic ester isapplied to the middle of the support; the support is subsequentlyrotated about the axis which runs through the point of application andis perpendicular to the plane of the support, so that a uniform liquidfilm is produced.

b) The polysilicic ester is sprayed in diluted or undiluted form ontothe support.

Further methods are, for example: application by means of a techniquesimilar to screen printing or by means of a roller.

The atmosphere in which the further hydrolysis and polymerization of thepolysilicic ester takes place comprises water vapour together witheither ammonia or hydrochloric acid. Such an atmosphere can begenerated, for example, by introducing the support to which the liquidpolysilicic ester has been applied into a chamber in which an openvessel of fuming hydrochloric acid is located and subsequentlyintroducing hot steam into the chamber.

The polymerization of the polysilicic esters preferably takes place atfrom 15 to 80° C., in particular from 40 to 60° C., and takes from 3minutes to 1 hour, depending on the temperature. After thepolymerization and after the silica layer has been washed, theseparation material is dried by customary methods at from 15 to 120° C.(depending on temperature, for from 5 to 30 minutes). The separationlayers obtained according to the invention have a thickness of from 2 to20 μm, in particular from 4 to 10 μm.

The mesopores formed in the process of the invention can be enlarged bycustomary methods, in particular by treatment with aqueous ammoniasolution.

The silica gel layer on the support can additionally be modified usingcustomary methods. For example, separation effectors such as ionicgroups for ion-exchange chromatography or hydrophobic groups forreversed-phase chromatography can be introduced. Such modificationmethods and suitable separation effectors are known to those skilled inthe art and are described in handbooks such as Packings and StationaryPhases in Chromatographic Techniques (K. K.: Unger ed.; Marcel Dekker,New York and Basel (1990)) or Porous Silica (K. K. Unger ed.; Elsevier,Amsterdam, Oxford New York (1979)). Particularly suitable modificationmethods for separation media for thin layer chromatography are thosedisclosed in DE 27 12 133 and DE 28 09 137.

Even without further explanations, a person skilled in the art will beable to make use of the above description in its widest scope. Thepreferred embodiments and examples are therefore to be regarded merelyas descriptive and do not constitute any limitation.

The full disclosure of all patent applications, patents and publicationsmentioned above and below, and also the corresponding application DE 19805 395.9, filed on Feb. 11, 1998, are incorporated by reference into thepresent application.

EXAMPLES PRODUCTION EXAMPLES Example 1

A 20×20 cm glass plate as is customary as support in thin layerchromatography is cleaned with sodium hydroxide solution andsubsequently with Extran® and finally with water. About 0.8 ml of a 50%(v:v) ethanolic solution of polyethoxysilane (mean molecular weightabout 1100) is subsequently sprayed on. The sprayed glass plate isintroduced into a closed chamber in which an open vessel of fuminghydrochloric acid is located, hot steam (4 bar) is introduced and theglass plate is treated in this way for 30 minutes at 60° C. The coatedglass plate is subsequently washed with water and dried. For use, theglass plate is cut into pieces of the desired size and shape.

The result is a separation material for thin layer chromatography whoseseparation medium layer consists of porous silica gel in a thickness ofabout 5 μm. The separation medium layer has a specific surface area of410 m²/g and a specific pore volume of about 0.32 ml/g (nitrogenadsorption by the BET method); this gives a mean pore size of 3 nm.

Example 2

A separation material produced as described in Example 1 is dipped intoa solution of methyloctadecyl-dichlorosilane (10% by weight in toluene)for 5 minutes. The separation material is subsequently rinsed by dippinginto various solvents: 5 minutes in toluene, 1 minute each indichloromethane/methanol (1:1; v:v), in acetone/water (1:1; v:v) and inmethanol. The plate is subsequently dried for 30 minutes at roomtemperature in the fume cupboard and for another 15 minutes at 120° C.in a drying oven.

This results in a C₁₈-modified separation material for reversed-phasechromatography.

Example 3

150 ml of fuming hydrochloric acid are placed in a conditioning chamber.The closed chamber is preheated to 60° C. (1 hour) in a drying oven. Thechamber is taken from the drying oven and steam is introduced into thechamber (shut-off valve opened slightly for about 5 seconds).

A washed glass plate (10×20 cm) is cleaned with ethanol and dried. 400μl of polyethoxysilane (mean molecular weight: 1000) are subsequentlyapplied to the glass plate and drawn off using a 12 μm roller. Thecoated glass plate is placed on an expanded polystyrene plate (20×20 cm)in the conditioning chamber and heated at 60° C. for 30 minutes. The TLCplate is subsequently washed with water and dried in air.

The silica gel layer has a thickness of 12+1 μm and adheres firmly tothe glass plate.

The silica gel layer can be modified as described in Example 2.

USE EXAMPLES Use Example A Separation of a Dye Mixture

Four spots of 50 nl each of a dye mixture comprising 0.1% by weight ofviolet I, 0.1% by weight of fast nitro blue 2B and 0.1% by weight ofSico oil-soluble blue 50401N dissolved in toluene are applied by meansof a Hamilton syringe to a 14×56 mm TLC plate produced as described inExample 1. As eluant, use is made of toluene in a normal chamber (50 mlglass beaker with clock glass as cover) without chamber saturation.After an elution time of 1 minute, sufficient separation has beenachieved; the elution distance is 8 mm.

FIG. 1 schematically shows the result of this experiment.

Use Example B

Chromatography of Cholesterol

50 nl of a solution of cholesterol (0.1% by weight indichloromethane/methanol (1:1; v:v)) are applied by means of a Hamiltonsyringe to a 14×56 m TLC plate produced as described in Example 2 andthe chromatogram is developed in acetone/water (95:5; v:v) (chambersaturation; elution distance: 7 mm; elution time: about 5 minutes).After the plate has been dried, it is sprayed with MnCl₂/sulfuric acidreagent and the colour is developed in a drying oven at 120° C. (5minutes) An hRf value of 28.6 was found; this value indicates that thesilica gel layer has been modified and the cholesterol is retained bythe stationary phase.

What is claimed is:
 1. Separation material for thin layer chromatographyconsisting of a support and a separation medium layer obtainable by thefollowing process steps: a) cleaning of the support; b) application of aliquid film comprising a polysilicic ester to a support; c) introductionof the support with the liquid film into an atmosphere which effectshydrolysis and further polymerization of the polysilicic ester; d)hydrolysis and further polymerization of the polysilicic ester atconstant temperature; e) washing of the silica layer; f) drying of thesilica layer.
 2. Separation material according to claim 1, furthercharacterized by modification of the silica gel surface by means ofseparation effectors.
 3. Process for producing a separation material forthin layer chromatography, which comprises the following steps: a)cleaning of the support; b) application of a liquid film comprising apolysilicic ester to a support; c) introduction of the support with theliquid film into an atmosphere which effects hydrolysis and furtherpolymerization of the polysilicic ester; d) hydrolysis and furtherpolymerization of the polysilicic ester at constant temperature; e)washing of the silica layer; f) drying of the silica layer.
 4. Processaccording to claim 3, wherein the said liquid film is applied byspraying on.
 5. Process according to claim 3, wherein the said liquidfilm is applied by firstly applying the liquid at one point andsubsequently rotating the support about the point of application, withthe axis of rotation being perpendicular to the plane of the support. 6.Process according to claim 3, wherein the said liquid film is applied bymeans of a roller.
 7. A method of separating at least two substances bythin layer chromatography comprising applying the at least two materialsto the separation materials of claim 1 and eluting the at least twosubstances.